The WHO estimates that in 2018, malaria caused ~228M clinical episodes and ~405,000 deaths worldwide. Despite an annual investment of >$3 billion for control measures, 2018 was the 3rd consecutive year in which there was no decrease in malaria cases, indicating a saturation of capacity to implement further impact with currently available strategies. There is an urgent unmet medical need for a highly efficacious malaria vaccine that prevents infection and disease. Sanaria?s 1st generation vaccine based on radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) PfSPZ Vaccine has been assessed in 19 clinical trials in 6 countries in Africa, 2 countries in Europe, and the US, and received Fast Track designation from the FDA. Clinical trials with Phase 3 compliant vaccine will begin in mid 2020, and licensure (marketing authorization) in the US (FDA) and in Europe (EMA) is planned for 2022. PfSPZ Vaccine is targeted to prevent malaria in travelers to and residents of Africa, and for immunizing the entire community to halt transmission and eliminate malaria from geographically focused areas of Africa. During the next 5-10 years, we aim to significantly increase potency and decrease cost of goods (COGs) of PfSPZ-based vaccines so they can be optimally used to prevent Pf malaria. One of our strategies to improve the breadth of protection in our vaccines is to include additional strains of Pf from geographically diverse regions such as Pf7G8 (Brazil) and PfNF135.C10 (Cambodia). Other than PfNF54 (West Africa strain), all other Pf strains assessed in humans are poor gametocyte producers. An approach to improving the efficiency of large-scale production of PfSPZs from different geographic regions and decreasing COGs would be generating increased numbers of fertile gametocytes per unit of culture for production of PfSPZ in mosquitoes. In nature, gametocytogenesis occurs only in a small subset of blood stage parasites due to epigenetic suppression of gametocytogenesis-related genes. Control of gametocyte commitment would provide a powerful tool for improving production of PfSPZ. A key molecule in this process is the master switch transcription factor, PfAP2-G, the expression of which correlates directly with the percent of gametocytes produced by a given Pf strain. Deletion of this gene completely abolishes gametocyte production. We propose to increase the gametocyte production capacity of Sanaria?s vaccine strains and decrease PfSPZ manufacturing COGs by over-expressing this gene by using CRISPR-Cas9 gene editing to modify the pairing elements (PE) in the 3?-UTR of pfap2-g. As an alternative, we will also relieve epigenetic silencing of PfAP2-G by replacing its promoter with the constitutive calmodulin promoter in a conditionally regulatable gametocyte induction (on/off) system we have developed. We will generate enhanced gametocyte-producing lines of Pf from different geographic regions, compare and evaluate clones with enhanced gametocytogenesis and select those with optimal in vitro growth that maintain high prevalence and intensities of infectious PfSPZs in aseptic mosquitoes.